Use of IL-12 antagonists in the treatment of rheumatoid arthritis

ABSTRACT

Method of treating autoimmune conditions are disclosed comprising administering to a mammalian subject IL-12 or an IL-12 antagonist. In certain preferred embodiments the autoimmune condition is one which is promoted by an increase in levels of IFN-γ or TNF-α. Suitable conditions for treatment include multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes melitis and autoimmune inflammatory eye disease.

This application is a divisional of application Ser. No. 08/560,943,filed Nov. 20, 1995, now abandoned; which is a file-wrapper-continuationof application Ser. No. 08/212,629, filed Mar. 14, 1994, now abandoned;all of which are incorporated by reference herein.

BACKGROUND OF THE INVENTION

Gamma interferon (IFN-γ) and tumor necrosis factor-alpha (INF-α) havebeen implicated in the development, exacerbation and/or recurrence ofnumerous autoimmune conditions. For example, both IFN-γ and TNF-α havebeen associated with the course of multiple sclerosis [Choflon et al.,Eur. Cytokine Netw. 3(6), 1992, pp. 523-531; Steinman, ScientificAmerican, September 1993, pp. 107-114; Hofman et al., J. Exp. Med. 170,1989, pp. 607-612; Panitch et al., Neurology, 37, 1987, pp. 1097-1102]and Type-I diabetes (insulin-dependent diabetes melitis, IDDM) [Castanoet al., Annu. Rev. Immunol. 8, 1990, pp. 647-679; Campbell et al., J.Clin. Invest. 87, 1991, pp. 739-742]. While TNF-α has been found topromote development of rheumatoid arthritis [Feldmann et al., Progressin Growth Factor Research, 4, 1992, pp. 247-255], administration ofIFN-γ has been linked to improvements in arthritic subjects [Veys etal., J. Rheumatology, 15(4), 1988, pp. 570-574]. Studies have alsodemonstrated the involvement of IFN-γ in the autoimmune diseasesprocesses associated with systemic lupus erythematosus (SLE) [Funauchiet al., Tohoku J. Exp. Med., 164, 1991, pp. 259-267; Bankhurst, J.Rheumatology, 14(supp. 13), 1987, pp. 63-67], autoimmune thyroiditis[Tang et al., Eur. J. Immunol. 23, 1993, pp. 275-278], and autoimmuneinflammatory eye disease (e.g., autoimmune uveoretinitis) [Charteris etal., Immunology 75, 1992, pp. 463-467]. Development of autoimmunepulmonary inflammation [Deguchi et al., Clin. Exp. Immunol. 85, 1991,pp. 392-395] and Guillain-Barre syndrome [Baron et al., Proc. Natl.Acad. Sci. USA 90, 1993, pp. 4414-4418] have also been tied to TNF-αactivity.

Interleukin-12 (IL-12) is a heterodimeric cytokine which was originallyidentified as a factor which induces IFN-γ from T cells and naturalkiller cells as set forth in PCT/US91/06332, published Apr. 2, 1992.PCT/US91/06332 refers to IL-12 as Natural Killer Cell Stimulating Factoror NKSF. EP 433827, published Jun. 26, 1991 discloses IL-12 as acytotoxic lymphocyte maturation factor (CLMF). IL-12 also stimulatesnatural killer cells in vitro by increasing their ability to lyse targetcells at a level comparable to that obtained with IFN-α and IL-2,well-known activators of natural killer cells' cytotoxic activity.Additional in vitro activities of IL-12 which have been identifiedinclude induction of TNF-α; induction of T cell proliferation as aco-stimulant; suppression of IL-2 induced proliferation of naturalkiller blasts; suppression of IL-2 induced proliferation of T cellreceptor-γδ-positive cells; promotion of Th1 T cell differentiation fromprogenitors; enhancement of Th1, but not Th2 proliferation; enhancementof T cell cytolytic activity; enhancement of cytotoxic lymphocytegeneration; enhancement of natural killer and natural killer blastcytolytic activity; ex vivo enhancement of natural killer activity inperipheral blood mononuclear cells of IL-2-treated patients; inductionof adhesion molecules on natural killer cells; induction of perforin andgranzyme B mRNAs in natural killer blasts; induction of IL-2 receptorsubunits (p55, p75) on natural killer cells; suppression of IgEsynthesis by IFN-γ-dependent and independent mechanisms; modulation of Tcell development in fetal thymic organ cultures; and synergy with kitligand to promote growth of myeloid and B cell progenitors. The known invivo activities of IL-12 include induction of IFN-γ; enhancement ofnatural killer cell activity in spleen, liver, lungs and peritonealcavity; enhancement of generation of allo-specific cytotoxiclymphocytes; induction of extramedullary hematopoiesis in mouse spleen;reversible suppression of hematopoiesis in bone marrow; reversibleinduction of anemia, lymphopenia, and neutropenia in mice; suppressionof anti-IgD induced IgE, IgG1, and IL4 expression; increased survival inSCID mice treated with Toxoplasma gondii; cure of leishmaniasis insusceptible strains of mice; decreased bioburden in cryptococcosesmodel; suppression of tumor growth; and promotion of immunity to tumorcells. IL-12 is also induced in vivo in the shwarzman reaction model ofseptic shock.

Although IL-12 can induce production of IFN-γ and TNF-α in vivo, therelationship of in vivo levels of IL-12 to autoimmune diseases which areaffected by levels of IFN-γ and TNF-α has not been established.Furthermore, the effects of administration of IL-12 or antagonists ofendogenous IL-12 (such as anti-IL-12 antibodies) on autoimmune diseasesassociated with induction of IFN-γ or TNF-α have not been examined.

SUMMARY OF THE INVENTION

The present invention provides methods of treating (e.g., curing,ameliorating, delaying or preventing onset of, preventing recurrence orrelapse of) autoimmune conditions or diseases. In preferred embodiments,the condition is one promoted by an increase in levels of a cytokineselected from the group consisting of TNF-α or IFN-γ. Such conditionsinclude, without limitation, those selected from the group consisting ofmultiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis,autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmunethyroiditis, insulin dependent diabetes melitis and autoimmuneinflammatory eye disease. Multiple sclerosis and insulin-dependentdiabetes melitis are particularly preferred conditions for treatment inaccordance with the present invention as described herein.

In certain embodiments the method of treatment of the present inventioncomprises administering to a mammalian subject a therapeuticallyeffective amount of an IL-12 antagonist, preferably an antibody or otherspecies which is immunoreactive with IL-12. in certain preferredembodiments, the IL-12 antagonist is administered in a dose of about0.05 to about 25 mg/kg, preferably of about 0.2 to about 2 mg/kg. Theantagonists can also be administered in combination with apharmaceutically acceptable carrier.

In other embodiments, the method of treatment of the present inventioncomprises administering to a mammalian subject a therapeuticallyeffective amount of IL-12. In certain embodiments, the IL-12 may beadministered in a dose of about 0.001 to about 1000 μg/kg, preferablyabout 0.01 to about 100 μg/kg. The IL-12 can also be administered incombination with a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 presents graphs of data relating to the adoptive transfer ofexperimental allergic encephalomyelitis (EAE) using lymph node andspleen cells stimulated in vitro with PLP and rmIL-12. Spleens and Lymphnodes were harvested from mice 10 days after immunization with PLP andstimulated in vitro with antigen alone (open symbols) or antigen and20ng/ml rmIL-12 (closed symbols) as described in materials and methods.Disease was transferred using 30×10⁶ cells. The results are presented asmean score for (a) lymph nodes (n=7) and (b) spleen cells (n=5). Thedata is representative of at least two separate experiments. See example1.

FIG. 2 presents graphs of data relating to IFN-γ and TNF-α productionfrom LNC stimulated in vitro with PLP and IL-12. LNC (2.5×10⁶/ml) fromPLP immunized mice were cultured with PLP alone, PLP and rmIL-12 (20ng/ml) or PLP, rmIL-12 and anti-IFN-γ (5 μg/ml) for 96 hours prior tocell transfer with 30×10⁶ cells. (a) IFN-γ and TNF-α measured by ELISAin the supernatants of pooled cultures. (b) Mean disease score after thetransfer of stimulated lymph node cells. n=3 for PLP alone and PLP+IL-12and n=4 for PLP+IL-12+anti-IFN-γ. See example 1.

FIG. 3 depicts graphs of data relating to the effects of In vivoadministration of IL-12 on the adoptive transfer of EAE using PLPstimulated LNC. LNC from PLP immunized mice were cultured in vitro withantigen as described in materials and methods and transferred to naivemice. rmIL-12 (0.3 μg/mouse) was administered on days 0, 1 and 2 aftercell transfer (closed circles) and mice monitored for signs of disease.Control mice received and equal volume of saline (open circles). (a)Mean clinical score following the transfer of 30×10⁶ LNC cells (n=5).(b) Mean clinical score following the transfer of 10×10⁶ LNC (n=4). FIG.3a is representative of three separate experiments. See example 1.

FIG. 4 depicts graphs of data relating to the effects of in vivoadministration of anti-IL-12 antibody on the adoptive transfer of EAEusing PLP stimulated LNC. LNC from PLP immunized mice were cultured invitro with antigen as described in materials and methods and 30×10⁶cells transferred to naive mice. Anti-IL-12 antibody (sheep anti-mousepolyclonal antibody, 200 μg mouse) was administered by intraperitonealinjection starting on the day of cell transfer (closed circles). Controlmice received an equivalent amount of sheep IgG (open circles). (a) Meanclinical score following administration of αIL-12 antibody everyotherday from day 0 to day 6. (b) Mean clinical score followingadministration of αIL-12 antibody everyother day from day 0 to day 12.(n=5-7). See example 1.

FIGS. 5 and 6 present graphs of data relating to disease incidence inNOD mice upon administration of IL-12. See example 2.

DETAILED DESCRIPTION

The present invention provides methods for treating autoimmuneconditions. “Autoimmune conditions” are those in which the subject's ownimmune system reacts against the subject's cells or tissues, resultingin damage to those cells or tissues. A particular autoimmune conditionis “promoted by an increase in levels of a cytokine” when a increase inserum or tissue levels of such cytokine can cause or contribute to thedevelopment or recurrence of, or to the acceleration of the onset of,such autoimmune condition. Autoimmune conditions which are promoted byan increase in levels of IFN-γ and/or TNF-α include, without limitation,multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis,autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmunethyroiditis, insulin dependent diabetes melitis and autoimmuneinflammatory eye disease.

“IL-12 antagonists” include (1) species that will bind IL-12 orbiologically active fragments thereof, and (2) species that willinterfere with the binding of IL-12 to receptors or other bindingproteins. Antagonists that bind IL-12 include, without limitation,antibodies (mono- or polyclonal) and fragments thereof (including F_(ab)fragments), chimeric antibodies and fragments thereof, lectins, IL-12receptors or fragments thereof, reactive peptides or fragments thereof,and organic small molecules designed to mimic the bioactivity of IL-12receptors. Antagonists that interfere with IL-12 binding include,without limitation, chemically or genetically modified peptides ofIL-12, subunits of IL-12 and fragments thereof, homopolymers of IL-12subunits and fragments thereof, and organic small molecules designed tomimic the bioactivity of IL-12. Preferably, antagonists that interferewith IL-12 binding interfere with its binding to receptors which induceIFN-γ or TNF-α, without inducing the same level of such factors as wouldbinding of IL-12 to the receptor.

IL-12 antagonists can be produced by methods well known to those skilledin the art. For example, monoclonal IL-12 antibodies can be produced bygeneration of antibody-producing hybridomas in accordance with knownmethods (see for example, Goding. 1983. Monoclonal antibodies:principles and paractice. Academic Press Inc., New York; Yokoyama. 1992.“Production of Monoclonal Antibodies” in Current Protocols inImmunology. Unit 2.5. Greene Publishing Assoc. and John Wiley & Sons).Polyclonal sera and antibodies to IL-12 can be produced by inoculationof a mammalian subject with IL-12 or fragments thereof in accordancewith known methods. Chizzonite et al., J. Immunol. 148, 1992, p. 3117,describes the identification and isolation of an IL-12 receptor.Fragments of antibodies, receptors or other reactive peptides can beproduced from the corresponding antibodies by cleavage of and collectionof the desired fragments in accordance with known methods (see forexample, Goding, supra; Andrew et al. 1992. “Fragmentation ofImmunoglobulins” in Current Protocols in Immunology. Unit 2.8. GreenePublishing Assoc. and John Wiley & Sons). Chimerci antibodies may alsobe produced in accordance with known methods.

In methods of the present invention using IL-12, any form of IL-12 maybe used, so long as that form of IL-12 is capable of treating thedesired autoimmune condition. For example, IL-12 may be in the form ofthe heterodimer comprised of a 40 kD subunit disulfide-bonded to a 35 kDsubunit. When IL-12 is a heterodimer, the 40 kD subunit has substantialhomology to the 40 kD subunit of human IL-12 as set forth inPCT/US91/06332 and is disulfide bonded to a 35 kD subunit havingsubstantial homology to the 35 kD subunit of human IL-12 as set forth inthat same PCT publication. “Substantial homology” means greater than 75%homology at the amino acid level, while retaining the ability to treatthe desired autoimmune condition in a mammalian subject. Another form ofIL-12 which may be used in the present invention is an IL-12 subunitcapable of treating the desired autoimmune condition in a mammaliansubject. Such an IL-12 40 kD subunit has substantial homology to thehuman IL-12 40 kD subunit disclosed in PCT/US91/06332, and such an IL-1235 kD subunit has substantial homology to the human IL-12 35 kD subunitdisclosed in such PCT publication. Fragments of the IL-12 subunits thatretain IL-12 biological activity are also be useful to treat autoimmuneconditions in mammalian subjects, in accordance with the presentinvention.

For use in the present invention, it is preferable to produce IL-12recombinantly, through expression of DNA sequences encoding one or bothof the IL-12 subunits in a suitable transformed host cell. For example,using known methods the DNA sequences encoding human IL-12 set forth inPCT/US91/06332 may be linked to an expression vector such as pED(Kaufman et al., Nucleic Acids Res. 19, 4484-4490(1991)). In such anexpression vector, sequences which optimize translation such as CCACC(Kozak, M., Nucleic Acids Res. 12, 857-871 (1984)) may be added 5′ tothe initiation codon using known methods. The expression vectorcontaining the IL-12 subunits may then be transformed into a host cell,and protein expression may be induced and maximized, to produceheterodimeric human IL-12. For production of heterodimeric IL-12,the DNAsequences encoding the IL-12 subunits may be present on differentexpression plasmids or present in tandem on a single expression plasmid.

When a subunit or fragment of IL-12 is used to practice the presentinvention, it may also be produced recombinantly using known methods.For example, the DNA sequence encoding the human IL-12 40 kD subunit setforth in PCT/US91/06332 may be linked to an expression vector,transformed into a host cell, and expression induced and maximized toproduce the human IL-12 40 kD subunit. Similarly, the DNA sequencesencoding the human IL-12 35 kD subunit as set forth in the PCTpublication may be linked to an expression vector, transformed into ahost cell, and expression induced and maximized to produce thecorresponding protein. Of course, degenerate DNA sequences encoding theIL-12 subunits may also be employed to produce IL-12 for use in thepresent invention, as can DNA sequences encoding allelic variants of theIL-12 subunits. Chemically or genetically modified forms of IL-12 andits subunits can also be made in accordance with the methods disclosedin the PCT publication.

Any suitable expression vector may be employed to produce IL-12 for usein the present invention. For mammalian expression, numerous expressionvectors are known in addition to the pED vector mentioned above, such aspEF-BOS (Mizushima et al., Nucleic Acids Res. 18, 5322 (1990)); pXM,pJL3 and pJL4 (Gough et al., EMBO J. 4, 645-653 (1985)); and pMT2(derived from pMT2-VWF, A.T.C.C. #67122; see PCT/US87/00033). Suitableexpression vectors for use in yeast, insect, and bacterial cells arealso known. Construction and use of such expression vectors is wellwithin the level of skill in the art.

Suitable host cells for recombinant production of IL-12 useful in thepresent invention include, for example, mammalian cells such as Chinesehamster ovary (CHO) cells, monkey COS cells, mouse 3T3 cells, mouse Lcells, myeloma cells such as NSO (Galfre and Milstein, Methods inEnzymology 73, 346 (1981)), baby hamster kidney cells, and the like.IL-12 may also be produced by transformation of yeast, insect, andbacterial cells with DNA sequences encoding the IL-12 subunits,induction and amplification of protein expression, using known methods.

Recombinantly produced IL-12 can be purified from culture medium or cellextracts by conventional purification techniques. Culture medium or cellextracts containing IL-12 may be concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. Following the concentrationstep, the concentrate can be applied to a purification matrix such as agel filtration medium. Alternatively, an anion exchange resin can beemployed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose or other types commonly employed in proteinpurification. Alternatively, a cation exchange step can be employed.Suitable cation exchangers include various insoluble matrices comprisingsulfopropyl or carboxymethyl groups. The purification of IL-12 fromculture supernatant may also include one or more column steps over suchaffinity resins as lectin-agarose, heparin-toyopearl® or Cibacrom blue3GA Sepharose®; or by hydrophobic interaction chromatography using suchresins as phenyl ether, butyl ether, or propyl ether; or byimmunoaffinity chromatography. Finally, one or more reverse-phase highperformance liquid chromatography (RP-HPLC) steps employing hydrophobicRP-HPLC media, e.g., silica gel having pendant methyl or other aliphaticgroups, can be employed to further purify IL-12 for use in the presentmethods and compositions. Some or all of the foregoing purificationsteps, in various combinations, can be employed to provide asubstantially homogeneous isolated recombinant protein. Purification ofIL-12 subunits or fragments for use in the present invention may differfrom the optimal protocol for purification of the heterodimeric protein.

Preferably, when human IL-12 is produced recombinantly as set forthabove, it may be purified by the following method. The cells in whichthe human IL-12 has been made may be removed from the conditioned mediumby filtration, and the conditioned medium is loaded onto Q-SepharoseFastFlow™ (available from Pharmacia) or an equivalent anion exchangemedium, which has been equilibrated in 10-30 mM Tris-HCl, pH 7.8-8.3.The column is then washed extensively with the same buffer followed by awash with 30-45 mM histidine, pH 5.1-5.8, followed by a wash with theoriginal equilibration buffer. The recombinant human IL-12 is elutedfrom the column with a buffer containing 20-50 mM Tris-HCl, pH 7.8-8.5,and 0.15 to 0.50 M NaCl. The eluted material is loaded onto CM-SepharoseFastFlow™ (available from Pharmacia) or equivalent cation exchangemedium which has been equilibrated in 20-50 mM MES, pH 5.7-6.4, andwashed extensively with the same buffer. The column is washed with abuffer containing 20-40 mM sodium phosphate, pH 6.8-7.5 and 0.2-0.5 MNaCl. The eluted material is concentrated using an Amicon™ S1Y30 orequivalent spiral cartridge membrane which has been washed andequilibrated in the elution buffer used in the CM-Seplarose FastFlow™column. The material is concentrated to approximately 5% of the columnvolume of the final chromatographic step, which is size exclusion usingS200 Sephacryl™ (available from Pharmacia) or an equivalent sizeexclusion resin. The size exclusion column is equilibrated and elutedwith phosphate buffered saline, pH 7.2-7, and the recombinant humanIL-12 peak is collected and filtered for use in the method of theinvention. Those of skill in the art of protein purification may usealternative purification methods to obtain recombinantly-produced humanIL-12 for use in the method of the invention.

IL-12 may be purified from culture medium or extracts of cells whichnaturally produce the protein and used in the present invention.Exemplary purification schemes for naturally produced IL-12 are setforth in PCT/US91/06332 and in EP 433827.

Pharmaceutical compositions containing an IL-12 antagonist or IL-12which are useful in practicing the methods of the present invention mayalso contain pharmaceutically acceptable carriers, diluents, fillers,salts, buffers, stabilizers and/or other materials well-known in theart. The term “pharmaceutically acceptable” means a material that doesnot interfere with the effectiveness of the biological activity of theactive ingredient(s) and that is not toxic to the host to which it isadministered. The characteristics of the carrier or other material willdepend on the route of administration.

It is currently contemplated that the various pharmaceuticalcompositions should contain about 0.1 micrograms to about 1 milligramper milliliter of the IL-12 antagonist or IL-12.

Administration can be carried out in a variety of conventional ways.Intraperitoneal injection is the preferred method of administration ofthe IL-12 antagonist or IL-12. Intravenous, cutaneous or sub-cutaneousinjection may also be employed. For injection, IL-12 antagonist or IL-12will preferably be administered in the form of pyrogen-free,parenterally acceptable aqueous solutions. The preparation of suchparenterally acceptable protein solutions, having due regard to pH,isotonicity, stability and the like, is within the skill of the art.

The amount of IL-12 antagonist or IL-12 used for treatment will dependupon the severity of the condition, the route of administration, thereactivity of the IL-12 antagonist with IL-12 or the activity of theIL-12, and ultimately will be decided by the treatment provider. Inpracticing the methods of treatment of this invention, a therapeuticallyeffective amount of an IL-12 antagonist or IL-12 is administered. Theterm “therapeutically effective amount” means the total amount of eachactive component of the method or composition that is sufficient to showa meaningful patient benefit (e.g., curing, ameliorating, delaying orpreventing onset of, preventing recurrence or relapse of). One commontechnique to determine a therapeutically effective amount for a givenpatient is to administer escalating doses periodically until ameaningful patient benefit is observed by the treatment provider. Whenapplied to an individual active ingredient, administered alone, the termrefers to that ingredient alone. When applied to a combination, the termrefers to combined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially orsimultaneously. A therapeutically effective dose of an IL-12 antagonistin this invention is contemplated to be in the range of about 0.05 mg/kgto about 25 mg/kg. A therapeutically effective dose of IL-12 in thisinvention is contemplated to be in the range of about 0.001 to about1000 μg/kg. The number of administrations may vary, depending on theindividual patient and the severity of the autoimmune condition.

The IL-12 antagonist or IL-12 used in practicing the present inventionmay be administered alone or combined with other therapies forautoimmune conditions, such as steroidal or other anti-inflammatorytherapies and administration of other cytokines.

The methods of the present invention are further described in thefollowing examples, which are intended to illustrate the inventionwithout limiting its scope.

EXAMPLE 1

Experimental allergic encephalomyelitis (EAE) is a T cell mediatedautoimmune disease of the central nervous system (CNS). Disease can beinduced in susceptible strains of mice by immunization with CNS myelinantigens or alternatively, disease can be passively transferred tosusceptible mice using antigen stimulated CD4+ T cells [Pettinelli, J.Immunol. 127, 1981, p. 1420]. EAE is widely recognized as an acceptableanimal model for multiple sclerosis in primates [Alvord et al. (eds.)1984. Experimental allergic encephalomyelitis—A useful model formultiple sclerosis. Alan R. Liss, New York]. The effects ofadministration of an IL-12 antagonist on induction of EAE following theadoptive transfer of lymphocytes from immunized mice restimulated invitro with a synthetic peptide of myelin proteolipid protein (PLP).

Adoptive Transfer of PLP Sensitized LNC

Female SJL/J mice (7-10 wks) were purchased from The Jackson Laboratory,housed 5 to a cage and fed standard rodent chow diet with water adlibitum. Mice were immunized in two sites on the flank with 150 μg ofmouse PLP peptide comprising residues 139-151 (provided by G Brown,Genetics Institute). PLP was administered in ²⁰⁰ μl of Complete Freundsadjuvant containing 2 mg/ml Mycobacteria Tuberculosis H37RA (Difco). Onthe day of immunization mice were injected intravenously with 0.75×10¹⁰Bordatella pertussis bacilli (Massachusetts Public Health Laboratories,Boston, Mass.). Ten days after immunization, spleens and lymph nodes(popliteal, axillary and brachial) were harvested and the cellsresuspended in RPMI-1640 containing 10% FBS (Hyclone), 5×10⁻⁵ M2-Mercaptoethanol, 100 μg/ml streptomycin and 100 U/ml penicillin. PLPwas added to the cultures at 2 μg/ml. After 96 hours, the cells wereharvested, washed twice and 30×10^(≢)cells (either LNC or spleen)injected i.p. into naive SJL/J mice.

Clinical Evaluation of Disease

Mice were observed for clinical signs of EAE and scored on a scale of 0to 3 as follows:

0.5—Distal limp tail

1.0—Complete limp tail

1.5—Limp tail and hind limb weakness (unsteady gait)

2.0—Partial hind limb paralysis

3.0—Complete bilateral hind limb paralysis

In Vitro Administration of IL-12 Prior to Cell Transfer

Recombinant murine IL-12 (20 ng/ml, rmIL-12, Genetics Institute) wasadded to the in vitro cultures of lymph node or spleen cells withantigen prior to cell transfer. After 96 hours the cells were washedtwice and 30×10⁶ cell transferred to naive SJL/J mice to determine theeffects of IL-12 on the subsequent course of disease.

In separate experiments, LNC were cultured with either antigen alone,antigen plus IL-12 (20 ng/ml) or antigen plus IL-12 plus a neutralizingantibody to IFN-γ (5 μg/ml from Endogen). At the end of the cultureperiod supernatants were collected (pooled from three flasks) and IFN-γand TNF-α measured by ELISA (from Genzyme). 30×10⁶ cells from each groupwere transferred to naive mice which were monitored for signs ofdisease.

In vivo Administration of IL-12 and Anti-IL-12 Antibody Following theTransfer of PLP Stimulated LNC

rmIL-12 (0.3 μg/mouse, 200 μl i.p.) was administered to mice followingthe transfer of either 30×10⁶ or 10×10⁶ PLP stimulated LNC. IL-12 wasadministered on days 0, 1 and 2 following cell transfer. Control micereceived an equal volume of vehicle alone. To determine if IL-12 isinvolved in the induction of disease following the transfer of PLPstimulated LNC, mice were treated with 200 μg of a sheep polyclonalantibody against murine IL-12 (200 μl i.p.) on alternate days for either6 or 12 days in total following cell transfer and the mice monitored forsigns of disease. Control mice received and equal amount of sheep IgG.The mice were monitored.

Effect of rmIL-12 on Restimulation of PLP Primed T Cells in vitro

LNC from mice immunized with PLP as described in methods were stimulatedin vitro with antigen in the absence or presence of rmIL-12 (20 ng/ml)for 96 hours after which time they were tested for their ability totransfer disease to naive SJL/J mice. Mice receiving LNC stimulated invitro with PLP alone developed clinical signs of disease between days 6and 8. All control mice reached scores of 2 or greater (7/7) with 4 outof 7 mice progressing to complete hind limb paralysis which lastedbetween 1 and 4 days (FIG. 1a). All the control mice had recovered byday 19. In contrast mice receiving cells cultured in vitro with PLP andIL-12 developed severe EAE with rapid onset of clinical signs (FIG. 1a).By day 6, 4 out 7 mice had clinical scores of 2 or greater and all micewent on to develop full hind limb paralysis by day 8. In this particularexperiment 5 out of 7 mice failed to recover from the paralysis.

Spleen cells from PLP immunized mice stimulated in vitro with antigenfor 96 hours in the absence or presence of rmIL-12 (20 ng/ml) were alsoexamined to determine whether they could transfer disease to naive SJL/Jmice. The severity of disease following the adoptive transfer of 30×10⁶PLP stimulated spleen cells was mild compared to that induced by anequivalent number of PLP stimulated LNC, with only 2 out of 5 micedeveloping complete hind limb paralysis and the remaining 3 micedisplaying only mild signs of disease (FIG. 1b). Similar to the resultsobserved with LNC, the addition of rmIL-12 (20 ng/ml) to the in vitroculture of spleen cells prior to transfer exacerbated subsequent disease(FIG. 1b). Mice receiving spleen cells stimulated with PLP and rmIL-12developed clinical signs of disease by day 6 and all progressed to fullhind limb paralysis by day 12. The mean duration of paralysis in thesemice was 5.4 days (range 2-8 days).

Cytokine Production Following in vitro Stimulation of LNC with PLP andIL12

To determine the effects of IL-12 on cytokine production during the invitro stimulation with antigen, LNC from PLP primed mice were culturedwith either PLP alone, PLP and IL-12 (20 ng/ml) or PLP, IL-12 and aneutralizing anti-IFN-γ antibody. At the end of the in vitro culture,IFN-γ and TNF-α in the supernatant were measured by ELISA and the cellstested for their ability to transfer disease to naive mice. The additionof IL-12 during the in vitro stimulation of LNC with PLP resulted in agreater than 10 fold increase in IFN-γ (5.2 ng/ml control and 64 ng/mlIL-12) and a two fold increase in TNF-α in the cell culture supernatant(FIG. 2a). The addition of a neutralizing antibody to IFN-65 during theculture of LNC with antigen and IL-12 completely blocked IFN-γdetection, but had no effect on the increase in TNF-α in thesupernatants which remained approximately two fold higher relative tocontrols (100 pg/ml controls compared to 180 pg/ml with αIFN-γantibody). Furthermore, transfer of the cells stimulated in vitro withPLP and IL-12 in the presence of a neutralizing antibody to IFN-γ werestill capable of inducing severe disease with the same kinetics andduration to that seen following the transfer of cells stimulated withPLP and IL-12 alone (FIG. 2b).

The Effect of in vivo Administration of IL-12 on Disease Progression

Following the transfer of 30×10⁶ PLP stimulated LNC mice wereadministered rmIL-12 (0.3 μg/mouse) or saline for 3 days and the effectson the subsequent course of disease monitored. The onset and progressionof disease in the controls was similar to that described above withclinical signs evident between days 6-8 after the transfer of LNC with80% of the mice progressing to full bilateral hind limb paralysis. Peakdisease in the control mice lasted approximately 3 days after which timethe mice spontaneously recovered (FIG. 3a). Administration of rmIL-12(0.3 μg/mouse) for 3 days after the transfer of an equivalent number ofprimed LNC from the same in vitro cultures dramatically altered thecourse of disease. Although the time of onset of symptoms was onlyslightly earlier in the IL-12 treated mice (day 5), the subsequentprogression to peak disease was accelerated with all mice displayingfull hind limb paralysis by day 8. The duration of paralysis was alsosignificantly prolonged lasting up to 14 days (range 11-14). Severalmice treated with rmIL-12 that developed prolonged paralysis whichpersisted after the controls had fully recovered were sacrificed.

In a separate experiment, the effects of in vivo administration ofrmIL-12 on disease severity was examined following the transfer of asuboptimal number of LNC (10×10⁶ cells). Control mice receiving thislower number of LNC developed mild disease (FIG. 3b) with 1 out of 4animals progressing to full hind limb paralysis and only minimal diseasein the remaining 3 controls. In contrast, mice treated with rmIL-12 invivo following the transfer of 10×10⁶ LNC cells developed full clinicalsymptoms of disease with all mice scoring 2 or greater and 3 out of 4mice progressing to full hind limb paralysis. The effects of rmIL-12were also apparent after the transfer of as few as 5×10⁶ LNC cells with3 out of 5 mice reaching a score of 1. At this cell number controlsshowed no signs of disease (data not shown).

The effects of Anti-IL-12 Antibody Administration on the Course ofDisease

To determine if endogenous IL-12 plays an essential role in diseasetransfer, mice were treated with 200 μg of a sheep polyclonal antibodyto murine IL-12 every other day for either 6 or 12 days following thetransfer of 30×10⁶ PLP stimulated LNC cells. Controls received an equalamount of sheep IgG. The onset of clinical signs in the Sheep IgGtreated controls was similar to that seen in untreated mice receivingPLP stimulated LNC (day 6-7, FIG. 4a). All control mice developed signsof disease graded 2 or greater (70% developed full paralysis).Administration of the ant-IL-12 antibody during the first 6 days aftertransfer did reduce the severity of disease, however, the onset ofclinical signs was delayed by approximately 7 days. These micesubsequently went on to develop disease with all mice reaching a scoreof 2 or greater (80% developed full paralysis) with a similar timecourse of recovery to control animals. To determine if this delay ofdisease transfer could be sustained by a longer administration ofanti-IL-12 antibody, we treated mice for 12 days following adoptivetransfer of PLP primed LNC. Mice treated with anti-EL-12 antibody everyother day for 12 days after the transfer of PLP stimulated LNC not onlyshowed a more sustained delay in the kinetics of disease onset but alsoexperienced dramatically reduced clinical disease with only 2 out of 5mice developing mild signs of disease (FIG. 4b).

EXAMPLE 2

NOD/LtJ mice (Jackson Laboratories) were treated with IL-12 to gauge theeffect of the cytokine on an accepted animal model of insulin-dependentdiabetes melitis (IDDM) [Kutani et al., Adv. Immunol. 51, 1992, p. 285].Female NOD mice spontaneously develop an IDDM-like disease withdestruction of the Beta cells in the pancreas and spilling of glucoseinto the urine beginning around 12-14 weeks of age. In the inventor'sanimal facility, female NOD mice show a disease incidence ofapproximately 88% by 30 weeks of age.

Female NOD mice were treated with two different protocols. In TreatmentA, mice were given 10, 1 or 0.1 μg (0.5, 0.05 or 0.005 mg/kg) murineIL-12 (mIL-12) i.p. three times a week for two weeks beginning at 9-11weeks of age. In Treatment B, mice were given 1 or 0.1 μg mIL-12 i.p.once a week beginning at 9 weeks of age and were continued on treatmentuntil 25 weeks of age.

Mice under Treatment A receiving all three doses showed statisticallysignificant decreases of disease incidence, with the 10 μg dose beingmost effective (17% disease incidence) (see Table 1 and FIG. 5). Miceunder Treatment B receiving 1 μg weekly showed a large decrease indisease incidence (20%), while mice receiving 0.1 μg did not show ameasurable change in disease incidence (80%) (see Table 1 and FIG. 6).

TABLE 1 INCIDENCE OF DIABETES IN IL-12 TREATED NOD MICE IL-12 3X/week 2weeks* IL-12 weekly† age unRx 10 μg 1 μg 0.1 μg 1 μg 0.1 μg 8 0/10 0/109 0/10 0/10 10  0/10 0/6 1/5  0/11 0/10 0/10 11  0/20 1/6 2/5  2/11 0/100/10 12  1/25 1/6 2/10 2/11 0/10 0/10 13  2/25 1/6 2/10 2/11 0/10 0/1014  3/25 2/6 2/10 2/11 0/10 1/10 15  4/25 1/6 2/10 3/11 0/10 1/10 16 5/25 1/6 2/10 3/11 0/10 4/10 17  6/25 1/6 3/10 3/11 0/10 4/10 18  6/251/6 3/10 4/11 0/10 7/10 19  9/25 1/6 3/10 4/11 1/10 7/10 20 12/25 1/64/10 5/11 1/10 7/10 21 12/25 1/6 4/10 5/11 1/10 7/10 22 14/25 1/6 5/105/11 1/10 7/10 23 15/25 1/6 5/10 5/11 1/10 7/10 24 16/25 1/6 5/10 5/111/10 7/10 25 18/25 1/6 5/10 5/11 1/10 7/10 26 20/25 1/6 5/10 5/11 2/107/10 27 20/25 1/6 5/10 5/11 2/10 7/10 28 21/25 1/6 5/10 5/11 2/10 7/1029 22/25 1/6 5/10 5/11 2/10 7/10 30 22/25 1/6 5/10 5/11 2/10 8/10*treatment started at 9-10 weeks of age and continued for two weeks†treatment started at 9 weeks of age and continued for 15 weeks

All patent and literature references cited herein are incorporated byreference as if fully set forth.

What is claimed is:
 1. A method for treating rheumatoid arthritis in ahuman subject, said method comprising administering to said subject atherapeutically effective amount of an IL-12 antagonist that binds withIL-12, wherein said antagonist is chosen from a group consisting of anantibody immunoreactive with IL-12 and an antibody fragmentimmunoreactive with IL-12.
 2. The method of claim 1, wherein saidantagonist is administered in combination with a pharmaceuticallyacceptable carrier.
 3. The method of claim 1, wherein said antagonist isan antibody immunoreactive with IL-12.
 4. The method of claim 1, whereinsaid antagonist is an antibody fragment is immunoreactive with IL-12. 5.The method of claim 1, wherein the antibody is a monoclonal antibody. 6.The method of claim 1, wherein the antibody binds to a heterodimercomprised of a 40 kD subunit and an 35 kD subunit of IL-12.
 7. Themethod of claim 1, wherein said antagonist is administered in a dose offrom about 0.05 to about 25 mg/kg.
 8. The method of claim 1, whereinsaid antagonist is administered in a dose of from about 0.2 to about 2mg/kg.
 9. The method of claim 1, wherein said antagonist is administeredintravenously.
 10. The method of claim 1, wherein said antagonist isadministered subcutaneously.
 11. The method of claim 1, wherein saidantagonist is administered cutaneously.
 12. The method of claim 1,wherein said antagonist is administered in combination with othertherapies for autoimmune conditions.
 13. The method of claim 12, whereinsaid therapies comprise steroidal or other anti-inflammatory therapies.